Organophosphorus (OP) insecticides, widely used in agriculture and public health, display a very wide range of acute toxicity levels and are a potential hazard to many individuals. Metabolism and biochemical protective mechanisms were studied during the original grant period. One of the most significant observations was the presence in the brain of phosphorothionate desulfuration (activation) activity which, while very low compared to liver, correlated directly with acute toxicity level, whereas hepatic activity did not. A second important observation was that the Ca++- dependent A-esterases hydrolyze only a few oxons at low, toxicologically-relevant substrate concentrations, and this hydrolysis contributes to the low toxicity displayed by their corresponding phosphorothionates. The following hypothesis will be investigated in the renewal project: The overall toxicity level of a phosphorothionate insecticide is determined mainly by the level of brain desulfuration activity, with lesser contributions by the detoxication pathways. This hypothesis will be investigated primarily by intensive study in the rat of the monooxygenases in the brain, and the A-esterases in several tissues, primarily the liver. There will be three major specific aims: 1) To characterize the brain monooxygenases responsible for phosphorothionate desulfuration through immunological recognition, substrate specificity and kinetics of brain cytochrome P450; 2) To characterize the A-esterases responsible for oxon hydrolysis by tissue and subcellular distribution and assessment of whether the low affinity and high affinity esterases are identical; and 3) A structure-activity relationship study to characterize the structural requirements of substrates for low brain phosphorothionate activation and for high affinity oxon hydrolysis. The resultant information will be of value in the rational development of safer OP insecticides.